1. Field of the Invention
The present invention is broadly concerned with improved immunoassays for the detection of antibodies against Lawsonia intracellularis, as well as an effective antigen comprising and preferably consisting essentially of an antigenic extract of L. intracellularis lipopolysaccharide. The preferred assay is an indirect-type ELISA assay having excellent specificity and sensitivity, allowing use of the assay for detection of low levels of antibody in animal-derived specimens during initial stages of infection prior to the onset of clinical signs of disease.
2. Description of the Prior Art
Porcine proliferative enteritis (PPE), known as ileitis, intestinal adenomatosis, or necrotic enteritis, is a naturally occurring disease that can affect pigs from waning to young adult stage. PPE was formerly believed to be caused by a camplyobacter-like organism or ileal symbiont intracellularis. More recently, it has been established that the causative agent is Lawsonia intracellularis, an obligate intracellular, gram-negative bacterium. The disease is of economic importance owing to death loss, increased medication costs, poor weight gain and decreased food conversion in affected animals.
A key element in the rational therapy and effective control of PPE is a rapid and accurate identification of etiologic agents. PPE may be diagnosed by observation of gross lesions and is confirmed by observation of specific hystopathological lesions in which the intracellular curved rods are demonstrated by special staining methods incorporating the use of an anti-Lawsonia monoclonal antibody. Ideally, a final determination should be made through the isolation of the causative agent. However, the isolation and culture of L. intracellularis requires specialized cell culture techniques.
Attempts have been made in the past to develop rapid assays for the detection of anti-Lawsonia antibodies. Current serological-based assays such as indirect fluorescence antibody test (IFAT) and immuno-peroxidase assay (IPMA) demonstrate good sensitivity and specificity in the detection of anti-Lawsonia antibodies antemortem in pig serum. Knittel J P, Jordan D M, Schwartz J K, et al. Evaluation of Antemorten Polymerase Chain Reaction and Serological Methods for Detection of Lawsonia Intracellularis-exposed Pigs. American Journal of Veterinarian Research. 59:722-726 (1998). Guedes R M C, Gebhart C J, Deen J, et al. Validation of an Immunopreoxidase Monolayer Assay as a Serological Test for Porcine Proliferative Enteropathy. Journal of Veterinarian Diagnostic Investigation. 14:528-530(2002), the teachings and content of these references are hereby incorporated by reference. However, neither of these assays is sensitive enough to detect the lower concentrations of anti-Lawsonia antibodies often found in pig serum during the initial exposure and onset of infection time periods. In addition, these prior assays rely on highly skilled technicians to accurately conduct the tests and interpret the results, e.g., the results are subjectively obtained by spending many hours looking into a microscope, analyzing wells illuminated by UV or natural light, searching for L. intracellularis or L. intracellularis-infected cells stained fluorescent green or red representing a “positive” sample.
Enzyme-linked immunoassays (ELISA) have also been developed for detection of anti-Lawsonia antibodies. These prior efforts failed to produce a sufficiently sensitive and specific assay owing to various limitations including poor antigen quality and quantity, variability in antibody titers, overlapping antibody titer between infected and non-infected pigs lack of a valid positive/negative “cut-off” value, and cross-reactivity of pig antibodies to non-specific antigen components. Holyoake P K, Cutler R S, Caple I W, Monckton R P. Enzyme-linked Immunosorbent Assay for Measuring Ileal Symbiont Intracellularis-specific Immunoglobulin G Response in Sera in Pigs. Journal of Clinical Microbiology. 31: 1980-1985 (1994), the teachings and content of which is hereby incorporated by reference.
There is accordingly a need in the art for an improved anti-Lawsonia antibody assay which is highly sensitive and specific, permitting accurate detection of low concentrations of antibodies in animal-derived specimens during the early stages of infection.
Lipopolysaccharides (LPS) are a major suprastructure of gram-negative bacteria such as L. intracellularis which contributes greatly to the structural integrity of the bacteria, and protects them from host immune defenses. LPS's form a part of the Gram negative bacteria cell wall and comprise three parts: polysaccharide side chains, core polysaccharides and lipid A. Lipid A may contain unusual fatty acids (e.g., hydroxy-mysteric acids) while core polysaccharides often contain unusual sugars (e.g., KDO, keto-deoxyoctulonate and heptulose). The polysaccharide side chain is referred to as the O-antigen of the bacteria. LPS's function as endotoxin, because they can bind to the CD14 receptor of macrophage, triggering the whole cascade for macrophage/endothelial cells to secrete pro-inflammatory cytokines.